DESCRIPTION (provided by applicant): The human genome contains at least 30,000 unique open reading frames that may yield >100,000 polypeptide products. These products assemble into more than a million biologically relevant structures or proteins of interest per organism. Crystallization and X-ray diffraction of these proteins is routinely performed to determine their 3D structure which needs to be understood for any effective protein engineering, rational drug design, or controlled drug delivery. Crystal structures of proteins such as enzymes, ribosomal proteins, bacterial toxins, hormones, and receptors are essential to understand important physiological processes and also to understand and treat diseases caused by microorganisms. Recent antiviral drugs against AIDS are enzyme inhibitors, and their design was based on the detailed protein-drug interactions provided by crystal structures of the enzymes with the drugs, based on structure-activity relationships.
In order to obtain a protein crystal, large amounts of protein need to be screened to find favorable conditions for crystallization but unfortunately many interesting proteins are very expensive or sometimes even difficult to express in large quantities. And in cases where native proteins are desirable to be crystallized, they could be available in extremely limited quantities. The equipment currently used for screening and optimization of crystallization conditions is very expensive and uses large amounts of precious proteins.
In phase I, we have successfully demonstrated a protein crystallization lab-on-a-chip using 25 nanoliter protein droplets of 3 proteins viz., lysozyme, glucose isomerase, and proteinase K. Such low volumes of protein per screening condition translate to significant cost savings in the upstream protein expression and purification. During phase I, we resolved a number of research issues to demonstrate the feasibility of performing crystallization screening on a digital microfluidic chip while in Phase II we will build on those results to: develop a screening chip that requires only 1 drop of protein to screen against 384 coarse grid reagents and develop an optimization chip which will programmably and automatically set up 96 fine grid conditions on-chip from just 5 reservoirs of salt, precipitant, buffer, water, and protein. Our instrument and disposable chips would be affordable even for an individual investigator.
Crystal structures of proteins such as enzymes, ribosomal proteins, bacterial toxins, hormones, and receptors are essential to understand important physiological processes. This structural information will help reveal the roles that proteins play in health and disease and will help rational design of new medicines. Recent antiviral drugs against AIDS are enzyme inhibitors, and their design was based on the detailed protein-drug interactions provided by crystal structures of the enzymes with the drugs, based on structure-activity relationships.